Raf1 fusions

ABSTRACT

The invention provides to RAF1 gene fusions, RAF1 fusion proteins, and fragments of those genes and polypeptides. The invention further provides methods of diagnosing and treating diseases or disorders associated with RAF1 fusions, such as conditions mediated by aberrant RAF1 expression or activity, or overexpression of RAF1.

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application No.62/010,242, filed Jun. 10, 2014, which is incorporated here by referencein its entirety to provide continuity of disclosure.

This invention relates to RAF1 (RAF proto-oncogeneserine/threonine-protein kinase, also known as proto-oncogene c-RAF, orc-Raf) gene fusions and RAF1 fusion proteins. The invention furtherrelates to methods of diagnosing and treating diseases or disordersassociated with RAF1 fusions, such as conditions mediated by aberrantRAF1 expression or activity, or conditions associated withoverexpression of RAF1.

Many forms of cancer are caused by genetic lesions that give rise totumor initiation and growth. Genetic lesions may include chromosomalaberrations, such as translocations, inversions, deletions, copy numberchanges, gene expression level changes, and somatic and germlinemutations. Indeed, the presence of such genomic aberrations is ahallmark feature of many cancers, including, for example, B cell cancer,lung cancer, breast cancer, ovarian cancer, pancreatic cancer, and coloncancer. In some models, cancer represents the phenotypic end-point ofmultiple genetic lesions that endow cells with a full range ofbiological properties required for tumorigenesis.

Recent efforts by The Cancer Genome Atlas (TCGA), the InternationalCancer Genome Consortium (ICGC), and dozens of other large-scaleprofiling efforts have generated an enormous amount of new sequencingdata for dozens of cancer types—this includes whole-genome DNA,whole-exome DNA, and full-transcriptome RNA sequencing. These effortshave led to the identification of new driver genes and fusion geneswithin multiple cancer types. Fusions, particularly fusions involvingkinases, are of particular interest, as such fusions have been shown tobe oncogenic, and have been successfully targeted by new therapeutics.For example, anaplastic lymphoma kinase (ALK), one of the receptortyrosine kinases, is known to become oncogenic when fused with variousgenes. See, e.g., M. Soda et al, “Identification of the transformingEML4-ALK fusion gene in non-small-cell lung cancer,” Nature 444:561-566(2007).

A need exists for identifying novel genetic lesions associated withcancer. For example, the presence of fusions involving a kinase insamples collected from more than one source can indicate that the kinaseis an oncogenic driver. The identification of such fusions can be aneffective approach to diagnosis of cancers and development of compounds,compositions, methods, and assays for evaluating and treating cancerpatients.

In one aspect, the invention provides methods for detecting the presenceof a RAF1 fusion in a biological sample; the methods include the stepsof: (a) obtaining a biological sample from a mammal; and (b) contactingthe sample with a reagent that detects a RAF1 fusion, to determinewhether a RAF1 fusion is present in the biological sample. In someembodiments, the sample can be from, e.g., a cancer patient. In someembodiments, the cancer is prostate adenocarcinoma. In some embodiments,the cancer is melanoma. In some embodiments, the fusion can be, e.g., anAGGF1:RAF1 fusion, an LMNA:RAF1 fusion, an MPRIP:RAF1 fusion, aPAPD7:RAF1 fusion, a CLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion. In someembodiments, the AGGF1:RAF1 fusion has all or a part of the nucleotideand/or amino acid sequence (such as, e.g., the fusion junction) setforth in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments,the LMNA:RAF1 fusion has all or part of the nucleotide and/or amino acidsequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:3and SEQ ID NO:4, respectively. In some embodiments, the MPRIP:RAF1fusion has all or part of the nucleotide and/or amino acid sequence(such as, e.g., the fusion junction) set forth in SEQ ID NO:5 and SEQ IDNO:6, respectively. In some embodiments, the PAPD7:RAF1 fusion has allor part of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.In some embodiments, the CLCN6:RAF1 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively. Insome embodiments, the TRAK1:RAF1 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively,

In another aspect, the invention provides methods of diagnosing apattern having a disease or disorder associated with aberrant RAF1expression or activity, or overexpression of RAF1; the methods include:(a) obtaining a biological sample from the patient; and (b) contactingthe sample with a reagent that detects a RAF1 fusion to determinewhether a RAF1 fusion is present in the biological sample, wherein thedetection of the RAF1 fusion indicates the presence of a disorderassociated with aberrant RAF1 expression or activity, or overexpressionof RAF1.

The invention also includes methods of determining a therapeutic regimenfor treating a cancer in a human subject; methods of identifying apatient likely to respond to treatment with a RAF1 inhibitor or a RAF1fusion inhibitor; methods of stratifying a patient population bydetecting a RAF1 fusion; methods of treating a patient; methods ofinhibiting the proliferation of cells containing a RAF1 fusion; methodsof reducing an activity of a RAF1 fusion; methods of treating acondition mediated by aberrant RAF1 expression or activity; methods oftreating a condition characterized by overexpression of RAF1; methods ofidentifying an agent that modulates the activity of a RAF1 fusion; andmethods of monitoring disease burden in a patient having a conditionmediated by RAF1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence of an AGGF1:RAF1 gene fusion (SEQID NO:1) comprising a portion of the AGGF1 gene (NM_018146) up to andincluding exon 5 (amino acid 290) and a portion of the RAF1 gene(NM_002880) starting at exon 8 (amino acid 279). The underlined codonsat nucleotides 868-870 and 871-873 encode the last amino acid of AGGF1and the first amino acid of RAF1, respective. The slash after nucleotide870 indicates the breakpoint (fusion junction) where translocation andin-frame fusion has occurred.

FIG. 2 depicts the amino acid sequence of an AGGF1:RAF1 fusion protein(SEQ ID NO:2). The slash between amino acids 290 and 291 indicates thebreakpoint or fusion junction between the AGGF1 and RAF1 proteins. Aminoacids 290-291 correspond to nucleotides 868-870 and 871-873 in SEQ IDNO:1.

FIG. 3 depicts the nucleotide sequence of an LMNA:RAF1 gene fusion (SEQID NO:3) comprising a portion of the LMNA gene (NM_170707) up to andincluding exon 10 (amino acid 566) and a portion of the RAF1 gene(NM_002880) starting at exon 8 (amino acid 279). The underlined codonsat nucleotides 1696-1698 and 1699-1701 encode the last amino acid ofLMNA and the first amino acid of RAF1, respectively. The slash afternucleotide 1698 indicates the breakpoint (fusion junction) wheretranslocation and in-frame fusion has occurred.

FIG. 4 depicts the amino acid sequence of an LMNA:RAF1 fusion protein(SEQ ID NO:4). The slash between amino acids 566 and 567 indicates thebreakpoint or fusion junction between the LMNA and RAF1 proteins. Aminoacids 566-567 correspond to nucleotides 1696-1698 and 1699-1671 in SEQID NO:3.

FIGS. 5A & 5B depict the nucleotide sequence of an MPRIP:RAF1 genefusion (SEQ ID NO:5) comprising an MPRIP gene (NM_201274) up to andincluding exon 22 (amino acid 1015) and a portion of the RAF1 gene(NM_002880) starting at exon 8 (amino acid 279). The underlined codonsat nucleotides 3043-3045 and 3046-3048 encode the last amino acid ofMPRIP and the first amino acid of RAF1, respectively. The slash betweennucleotides 3045 and 3046 indicates the breakpoint or fusion junctionwhere translocation and in-frame fusion has occurred.

FIG. 6 depicts the amino acid sequence of an MPRIP:RAF1 fusion protein(SEQ ID NO:6). The slash between amino acids 1015 and 1016 representsthe location where the two proteins are fused and corresponds tonucleotides 3043-3045 and 3046-3048 of SEQ ID NO:5.

FIG. 7 depicts the nucleotide sequence of a PAPD7:RAF1 gene fusion (SEQID NO:7) comprising a PAPD7 gene (NM_006999) up to and including exon 11(amino acid 423) and a portion of the RAF1 gene (NM_002880) starting atexon 10 (amino acid 331). The underlined codons at nucleotides 1267-1269and 1270-1272 encode the last amino acid of PAPD7 and the first aminoacid of RAF1, respectively. The slash between nucleotides 1269 and 1270indicates the breakpoint or fusion junction where translocation andin-frame fusion has occurred.

FIG. 8 depicts the amino acid sequence of a PAPD7:RAF1 fusion protein(SEQ ID NO:8). The slash between amino acids 423 and 424 represents thelocation where the two proteins are fused and corresponds to nucleotides1267-1269 and 1270-1272 of SEQ ID NO:7.

FIG. 9 depicts the nucleotide sequence of a CLCN6:RAF1 gene fusion (SEQID NO:9) comprising a CLCN6 gene (NM_001286) up to and including exon 2(amino acid 49) and a portion of the RAF1 gene (NM_002880) starting atexon 8 (amino acid 279). The underlined codons at nucleotides 145-147and 148-150 encode the last amino acid of CLCN6 and the first amino acidof RAF1, respectively. The slash between nucleotides 147 and 148indicates the breakpoint or fusion junction where translocation andin-frame fusion has occurred.

FIG. 10 depicts the amino acid sequence of a CLCN6:RAF1 fusion protein(SEQ ID NO:10). The slash between amino acids 49 and 50 represents thelocation where the two proteins are fused and corresponds to nucleotides145-147 and 148-150 of SEQ ID NO:9.

FIG. 11 depicts the nucleotide sequence of a TRAK1:RAF1 gene fusion (SEQID NO:11) comprising a TRAK1 gene (NM_001042646) up to and includingexon 9 (amino acid 325) and a portion of the RAF1 gene (NM_002880)starting at exon 8 (amino acid 279). The underlined codons atnucleotides 973-975 and 976-978 encode the last amino acid of TRAK1 andthe first amino acid of RAF1, respectively. The slash betweennucleotides 975 and 976 indicates the breakpoint or fusion junctionwhere translocation and in-frame fusion has occurred.

FIG. 12 depicts the amino acid sequence of an TRAK1:RAF1 fusion protein(SEQ ID NO:12). The slash between amino acids 325 and 326 represents thelocation where the two proteins are fused and corresponds to nucleotides973-975 and 976-978 of SEQ ID NO:11.

EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention is based, at least in part, on the discovery of novelrecombination or translocation events in cancer patients that result inat least a fragment of a RAF1 gene linked to a non-homologous promotervia a recombination or translocation event that may result in aberrantexpression (e.g., in a location where the kinase is not typicallyexpressed) or overexpression of the kinase domain of the RAF1 gene andthus, an increase in kinase activity. Thus, a new patient population isidentified, which is characterized by the presence of a RAF1 fusion,e.g., a RAF1 gene fusion or fusion protein. This new patient populationsuffers from or is susceptible to disorders mediated by aberrant RAF1expression or activity, or overexpression of RAF1, such as, e.g., acancer. In another aspect of the invention, a new subtype of cancer isidentified, which is characterized by the presence of the RAF1 fusionsdescribed herein. In some embodiments, the new patient populationsuffers from or is susceptible to prostate adenocarcinoma or melanomacharacterized by the presence of a RAF1 fusion. New methods ofdiagnosing and treating the patient population and the RAF1 fusioncancer subtype are also provided.

The term “RAF1 fusion” is used generically herein, and includes anyfusion molecule (e.g., gene, gene product (e.g., cDNA, mRNA, orprotein), and variants thereof) that includes a fragment of RAF1,particularly the coding region for the kinase domain of RAF1, and thecoding region of a second, non-homologous gene and a promoter sequencefrom the non-homologous gene, such that the coding sequence for thekinase domain of RAF1 is under control of the promoter of thenon-homologous gene. A RAF1 fusion protein generally includes the kinasedomain of RAF1.

RAF1 Gene Fusions and Fusion Proteins

RAF1 is a member of the Raf kinase family of serine/threonine-specificprotein kinases, from the TKL (Tyrosine-kinase-like) group of kinases.RAF1 is a MAP kinase (MAP3K) that can initiate the entire kinasecascade. Normal, cellular Raf genes have been shown to imitate to becomeoncogenes, by over stimulation of MEK1/2 and ERK1/2 activity.

RAF1 gene fusions are generated by a fusion between at least a part ofthe RAF1 gene and a part of another gene as a result of a translocation(including inversion) within a chromosome or between chromosomes. As aresult of a translocation, the RAF1 gene maybe placed under thetranscriptional control of the partner gene promoter, resulting inaberrant RAF1 expression or activity, or overexpression of RAF1. Theoverexpression can lead to certain cancers. Alternatively oradditionally, the partner gene may include a dimerization domain thatcauses RAF1 to become constitutively activated, or the fusion event maydelete an autoregulatory region of RAF1 leading to a constitutivelyactivated kinase. As used herein, the 5′-region is upstream of, and the3′-region is downstream of, a fusion junction or breakpoint in one ofthe component genes. RAF1 and the gene or protein that it is fused to isreferred to as “fusion partners.” Alternatively, they may be identifiedas a “RAF1 gene fusion” or a “RAF1 fusion protein” which arecollectively termed “RAF1 fusions.” The RAF1 fusions disclosed hereinhave a kinase activity. The phrase “having a kinase activity” as used inthis application means having an activity as an enzyme phosphorylatingthe side chain of an amino acid, such as serine or threonine.

In some exemplary embodiments, the fusion partner is all or a portion ofAGGF1 (Angiogenic factor with G patch and FHA domains 1). In otherexemplary embodiments, the fusion partner is all or a portion of LMNA(Lamin A/C). In other exemplary embodiments, the fusion partner is allor a portion of MPRIP (Myosin Phosphatase Rho Interacting Protein). Incertain exemplary embodiments, the fusion partner is all or a portion ofPAPD7 (PAP Associated Domain Containing 7). In other exemplaryembodiments, the fusion partner is all or a portion of CLCN6 (chloridetransport protein 6). In yet other exemplary embodiments, the fusionpartner is all or a portion of TRAK1 (trafficking protein, kinesinbinding 1).

Reference to “all or a portion” or “all or part” of a RAF1 gene fusionor SEQ NO:1, 3, 5, 7, 9, 11, or 13, means that the nucleotide sequencecomprises the entire RAF1 gene fusion nucleotide sequence or a fragmentof that sequence that comprises the fusion junction or breakpointbetween RAF1 and its fusion partner (such as, e.g., AGGF1, LMNA, MPRIP,PAPD7, CLCN6, or TRAK1). The fragment may comprise 7, 8, 9, 10, 12, 14,16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 150, 175, 200, 250, 300, or more nucleotidesspanning the fusion junction of the RAF1 gene fusion. Reference to “allor a portion” or “all or part” of a RAF1 fusion protein or SEQ ID NO:2,4, 6, 8, 10, 12, or 14, means an amino acid sequence that comprises theentire RAF1 fusion protein amino acid sequence or a fragment of thatsequence that comprises the fusion junction or breakpoint between RAF1and its fusion partner (such as, e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6,or TRAK1). The fragment may comprise 8, 10, 12, 14, 15, 16, 18, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more aminoacids spanning the fusion junction.

In certain embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the AGGF1 gene (e.g., an AGGF1 promotor ora functional fragment thereof, and one or more exons encoding AGGF1 or afragment thereof) and an exon of the RAF1 gene (e.g., one or more exonsencoding a RAF1 kinase domain or a functional fragment thereof). Such afusion can be referred to as an AGGF1:RAF1 fusion. In one embodiment,the AGGF1:RAF1 fusion comprises sufficient RAF1 sequence to driveexpression of a fusion protein that has kinase activity, e.g., haselevated activity as compared with wild type RAF1 in the same tissue orcell.

In some embodiments, the invention provides an AGGF1:RAF1 gene fusioncomprising the nucleotide sequence depicted in FIG. 1 (SEQ ID NO:1), ora fragment thereof that includes the fusion junction. SEQ ID NO:1comprises AGGF1 (NM_018046) up to oxen 5 (amino acid number 290) fusedto RAF1 (NM_002880), beginning at exon 8 (amino acid number 279). Insome embodiments the AGGF1:RAF1 gene fusion comprises a nucleotidesequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to all or part of SEQ ID NO:1.In some embodiments, the AGGF1:RAF1 gene fusion encodes a protein havingthe sequence depicted in FIG. 2 (SEQ ID NO:2) or a sequence that is atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% identical to all or part of SEQ ID NO:2.

In other embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the LMNA gene (e.g., an LMNA promoter or afunctional fragment thereof, and one or more axons encoding an LMNA or afragment thereof) and an exon of the RAF1 gene (e.g., one or more exonsencoding a RAF1 kinase domain or a functional fragment thereof). Such afusion can be referred to as an LMNA:RAF1 fusion. In one embodiment, theLMNA:RAF1 fusion comprises sufficient RAF1 sequence to drive expressionof a fusion protein that has kinase activity, e.g., has elevatedactivity as compared with wild type RAF1 in the same tissue or cell.

In some embodiments, the invention provides an LMNA:RAF1 gene fusioncomprising the nucleotide sequence depicted in FIG. 3 (SEQ ID NO:3), ora fragment thereof that includes the fusion junction. SEQ ID NO:3comprises LMNA (NM_170707) up to exon 10 (amino acid number 566) fusedto RAF1 (NM_002880), beginning at exon 8 (amino acid number 279). Insome embodiments the LMNA:RAF1 gene fusion comprises a nucleotidesequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to all or part of SEQ ID NO:3.In some embodiments, the LMNA:RAF1 gene fusion encodes a protein havingthe sequence depicted in FIG. 4 (SEQ ID NO:4) or a sequence that is atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% identical to all or part of SEQ ID NO:4.

In certain embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the MPRIP gene (e.g., an MPRIP promotor ora functional fragment thereof, and one or more exons encoding an MPRIPor a fragment thereof) and an exon of the RAF1 gene (e.g., one or moreexons encoding a RAF1 kinase domain or a functional fragment thereof).Such a fusion can be referred to as an MPRIP:RAF1 fusion. In someembodiments, the MPRIP:RAF1 fusion comprises sufficient RAF1 sequence todrive expression of a fusion protein that has kinase activity, e.g., haselevated activity as compared with wild type RAF1 in the same tissue orcell.

In other embodiments, the MPRIP:RAF1 fusion has the nucleotide sequencedepicted in FIG. 5 (SEQ ID NO:5), or a fragment thereof that includesthe fusion junction. SEQ ID NO:5 comprises MPRIP (NM_201274) up to andincluding exon 22 (amino acid number 1015) fused to RAF1 (NM_002880),beginning at exon 8 (amino acid 279). In some embodiments the MPRIP:RAF1gene fusion comprises a nucleotide sequence that is at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to all or part of SEQ ID NO:5. In sonic embodiments, theMPRIP:RAF1 fusion encodes a protein having the sequence depicted in FIG.6 (SEQ ID NO:6) or a sequence that is at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% identical to allor part of SEQ ID NO:6.

In certain embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the PAPD7 gene (e.g., a PAPD7 promotor ora functional fragment thereof, and one or more exons encoding a PAPD7 ora fragment thereof) and an exon of the RAF1 gene (e.g., one or moreexons encoding a RAF1 kinase domain or a functional fragment thereof).Such a fusion can be referred to as a PAPD7:RAF1 fusion. In oneembodiment, the PAPD7:RAF1 fusion comprises sufficient RAF1 sequence todrive expression of a fusion protein that has kinase activity, e.g., haselevated activity as compared with wild type RAF1 in the same tissue orcell.

In some embodiments, the invention provides a PAPD7:RAF1 gene fusioncomprising the nucleotide sequence depicted in FIG. 7 (SEQ ID NO:'7), ora fragment of thereof that includes the fusion junction. SEQ ID NO:7comprises PAPD7 (NM_006999) up to exon 11 (amino acid number 423) fusedto RAF1 (NM_002880), beginning at exon 10 (amino acid number 331). Insome embodiments the PAPD7:RAF1 gene fusion comprises a nucleotidesequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to all or part of SEQ ID NO:7.In some embodiments, the PAPD7:RAF1 gene fusion encodes a protein havingthe sequence depicted in FIG. 8 (SEQ ID NO:8) or a sequence that is atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% identical to all or part of SEQ ID NO:8.

In certain embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the CLCN6 gene (e.g., a CLCN6 promotor ora functional fragment thereof, and one or more exons encoding a CLCN6 ora fragment thereof) and an exon of the RAF1 gene (e.g., one or moreexons encoding a RAF1 kinase domain or a functional fragment thereof).Such a fusion can be referred to as a CLCN6:RAF1 fusion. In oneembodiment, the CLCN6:RAF1 fusion comprises sufficient RAF1 sequence todrive expression of a fusion protein that has kinase activity, e.g., haselevated activity as compared with wild type RAF1 in the same tissue orcell.

In some embodiments, the invention provides a CLCN6:RAF1 gene fusioncomprising the nucleotide sequence depicted in FIG. 9 (SEQ ID NO:9), ora fragment of thereof that includes the fusion junction. SEQ ID NO:9comprises the CLCN6 gene (NM_001286) up to and including exon 2 fused toRAF1 (NM_002880), beginning at exon 8. In some embodiments theCLCN6:RAF1 gene fusion comprises a nucleotide sequence that is at least85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least99% identical to all or part of SEQ ID NO:9. In some embodiments, theCLCN6:RAF1 gene fusion encodes a protein having the sequence depicted inFIG. 10 (SEQ ID NO:10) or a sequence that is at least 85%, at least 90%,at least 95%, at least 97%, at least 98%, or at least 99% identical toall or part of SEQ ID NO:10.

In certain embodiments of the invention, a fusion includes an in-framefusion of all or a portion of the TRAK1 gene (e.g., a TRAK1 promotor ora functional fragment thereof, and one or more exons encoding a TRAK1 ora fragment thereof) and an exon of the RAF1 gene (e.g., one or moreexons encoding a RAF1 kinase domain or a functional fragment thereof).Such a fusion can be referred to as a TRAK1:RAF1 fusion. In oneembodiment, the TRAK1:RAF1 fusion comprises sufficient RAF1 sequence todrive expression of a fusion protein that has kinase activity, e.g., haselevated activity as compared with wild type RAF1 in the same tissue orcell.

In some, embodiments, the invention provides a TRAK1:RAF1 gene fusioncomprising the nucleotide sequence depicted in FIG. 11 (SEQ NO:11), or afragment of thereof that includes the fusion junction. SEQ ID NO:11comprises the TRAK1 gene (NM_001042646) up to and including exon 9 fusedto RAF1 (NM_002880), beginning at exon 8. In some embodiments theTRAK1:RAF1 gene fusion comprises a nucleotide sequence that is at least85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least99% identical to SEQ ID NO:11. In some embodiments, the TRAK1:RAF1 genefusion encodes a protein having the sequence depicted in FIG. 12 (SEQ IDNO:12) or a sequence that is at least 85%, at least 90%, at least 95%,at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12.

The nucleic acid sequences of RAF1 gene fusions may be used as probes,primers, or bait to identify nucleotides from a biological sample thatinclude, flank, or hybridize to, RAF1 fusions, such as AGGF1:RAF1 (e.g.,all or part of SEQ ID NO: 1), LMNA:RAF1 (e.g., all or part of SEQ IDNO:3), MPRIP:RAF1 (e.g., all or part of SEQ ID NO:5), CLCN6:RAF1 (e.g.,all or part of SEQ ID NO:9), or TRAK1:RAF1 (e.g., all or part of SEQ IDNO:11) at, e.g., the fusion junctions. In certain embodiments, theprobe, primer, or bait molecule is an oligonucleotide that allowscapture, detection, and/or isolation of a RAF1 gene fusion in abiological sample. In certain embodiments, the probes or primers derivedfrom the nucleic acid sequences of RAF1 gene fusions (e.g., from thefusion junctions) may be used, for example, for polymerase chainreaction (PCR) amplification. The oligonucleotide can comprise anucleotide sequence substantially complementary to a fragment of theRAF1 gene fusion nucleic acid molecules described herein. The sequenceidentity between the nucleic acid fragment, e.g., the oligonucleotideand the target RAF1 gene fusion sequence, need not be exact, so long asthe sequences are sufficiently complementary to allow the capture,detection, and/or isolation of the target sequence. In one embodiment,the nucleic acid fragment is a probe or primer that includes anoligonucleotide between about 5 and 25, e.g., between 10 and 20, or 10and 15 nucleotides in length that includes the fusion junction of a RAF1fusion, such as e.g., AGGF1:RAF1 (e.g., all or part of SEQ ID NO: 1),LMNA:RAF1 (e.g. all or part of SEQ ID NO:3), MPRIP:RAF1 (e.g., all orpart of SEQ ID NO:5), PAPD7:RAF1 (e.g., all or part of SEQ ID NO:7,CLCN6:RAF1 (e.g., all or part of SEQ ID NO:9), or TRAK1:RAF1 (e.g., allor part of SEQ ID NO:11). In other embodiments, the nucleic acidfragment is a bait that includes an oligonucleotide between about 100 to300 nucleotides, 130 and 230 nucleotides, or 150 and 200 nucleotides inlength that includes the fusion junction of a RAF1 fusion, such as e.g.,AGGF1:RAF1 (e.g., all or part of SEQ ID NO: 1), LMNA:RAF1 (e.g., all orpart of SEQ ID NO:3), MPRIP:RAF1 (e.g., all or part of SEQ ID NO:5),PAPD7:RAF1 (e.g., all or part of SEQ ID NO:7), CLCN6:RAF1 (e.g., all orpart of SEQ ID NO:9), or TRAK1:RAF1 (e.g., all or part of SEQ ID NO:11).

In certain embodiments, the nucleic acid fragments hybridize to anucleotide sequence that includes a breakpoint or fusion junction, e.g.,a breakpoint or fusion junction as identified by a slash (“/”) in FIGS.1, 3, 5, 7, 9, or 11. For example, the nucleic acid fragment canhybridize to a nucleotide sequence that includes the fusion junctionbetween the AGGF1 transcript and the RAF1 transcript (e.g., nucleotides868-873 of SEQ ID NO:1), between the MPRIP transcript and the RAF1transcript (e.g., nucleotides 1696-1701 of SEQ ID NO:3), between theLMNA transcript and the RAF1 transcript (e.g., nucleotides 3043-3048 ofSEQ ID NO:5), between the PAPD7 transcript and the RAF1 transcript(e.g., nucleotides 1267-1272 of SEQ ID NO:7), between the CLCN6transcript and the RAF1 transcript (e.g., nucleotides 145-150 of SEQ IDNO:9), or between the TRAK1 transcript and the RAF1 transcript (e.g.,nucleotides 973-978 of SEQ ID NO:11), i.e., a nucleotide sequence thatincludes a portion of SEQ ID NO: 1, 3, 5, 7, 9, or 11. Examples includea nucleotide sequence within exons 1 to 5 of an AGGF1 gene and exons8-17 of a RAF1 gene (e.g., a portion of SEQ ID NO:1 comprisingnucleotides 868-873, 866-875, 861-880, 846-895, or 821-920); anucleotide sequence within exons 1-10 of an LMNA gene and exons 8 to 17of a RAF1 gene (e.g., a portion of SEQ ID NO:3 comprising nucleotides1696-1701, 1693-1702, 1688-1707, 1673-1722, or 1648-1747); a nucleotidesequence within exons 1-22 of an MPRIP gene and exons 8 to 17 of a RAF1gene (e.g., a portion of SEQ ID NO:5 comprising nucleotides 3043-3048,3041-3050, 3036-3055, 3021-3070, or 2996-3095); a nucleotide sequencewithin exons 1-11 of a PAPD7 gene and exons 10 to 17 of a RAF1 gene(e.g., a portion of SEQ ID NO:7 comprising nucleotides 1267-1272,1265-1274, 1260-1279, 1245-1294); a nucleotide sequence within exons 1-2of a CLCN6 gene and exons 8 to 17 of a RAF1 gene (e.g., a portion of SEQID NO:9 comprising nucleotides 145-150, 143-152, 138-157, 123-172, or98-197); or a nucleotide sequence within exons 1-9 of a TRAK1 gene andexons 8 to 17 of a RAF1 gene (e.g., a portion of SEQ ID NO:11 comprisingnucleotides 973-978, 971-980, 966-975, 951-990, or 926-1015).

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to a RAF1 gene fusionnucleic acid molecule described herein, and thereby allows thedetection, capture, and/or isolation of the nucleic acid molecule. Inone embodiment, a bait is suitable fur solution phase hybridization. Inother embodiments, a bait includes a binding entity or detection entity,e.g., an affinity tag or fluorescent label, that allows detection,capture, and/or separation, e.g., by binding to a binding entity, of ahybrid formed by a bait and a nucleic acid hybridized to the bait.

In exemplary embodiments, the nucleic acid fragments hybridize to anucleotide sequence that includes a fusion junction between the AGGF1transcript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:1 comprising nucleotides 868-873 (such as, e.g., a sequencecomprising nucleotides 866-875, 861-880, 846-895, or 821-920 of SEQ IDNO:1).

In other exemplary embodiments, the nucleic acid fragments hybridize toa nucleotide sequence that includes a fusion junction between the LMNAtranscript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:3 comprising nucleotides 1696-1701 (such as, e.g., a sequencecomprising nucleotides 1693-1702, 1688-1707, 1673-1722, or 1648-1747 ofSEQ ID NO:3).

In other exemplary embodiments, the nucleic acid fragments hybridize toa nucleotide sequence that includes a fusion junction between the MPRIPtranscript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:5 comprising nucleotides 3043-3048 (such as, e.g., a sequencecomprising nucleotides 3041-3050, 3036-3055, 3021-3070, or 2996-3095 ofSEQ ID NO:5).

In other exemplary embodiments, the nucleic acid fragments hybridize toa nucleotide sequences that includes a fusion junction between the PAPD7transcript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:7 comprising nucleotides 1267-1272 (such as, e.g., a sequencecomprising nucleotides 1265-1274, 1260-1279, 1245-1294, or 1219-1318 ofSEQ ID NO:7).

In some exemplary embodiments, the nucleic acid fragments hybridize to anucleotide sequence that includes a fusion junction between the CLCN6transcript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:9 comprising nucleotides 145-150 (such as, e.g., a sequencecomprising nucleotides 143-152, 138-157, 123-172, or 98-197 of SEQ IDNO:9).

In other exemplary embodiments, the nucleic acid fragments hybridize toa nucleotide sequence that includes a fusion junction between the TRAK1transcript and the RAF1 transcript, e.g., a nucleotide sequence withinSEQ ID NO:11 comprising nucleotides 973-978 (such as, e.g., a sequencecomprising nucleotides 971-980, 966-975, 951-990, or 926-1015 of SEQ IDNO:11).

Another aspect of the invention provides RAF1 fusion proteins (such as,e.g., a purified or isolated AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1 fusion protein), biologicallyactive or antigenic fragments thereof, and use of those polypeptides fordetecting and/or modulating the biological activity (such as tumorigenicactivity) of a RAF1 fusion protein. Exemplary embodiments of the RAF1fusion proteins comprise the amino acid sequence set forth in SEQ IDNO:2, 4, 6, 8, 10, or 12, and fragments of those sequences.

In some embodiments, the RAF1 fusion protein of the invention caninclude a fragment of an AGGF1 protein, an LMNA protein, an MPRIPprotein, a PAPD7 protein, a CLCN6 protein, or a TRAK1 protein, and afragment of a RAF1 protein. In one embodiment, the RAF1 fusion proteinis AGGF1:RAF1 fusion protein having the amino acid sequence of SEQ IDNO:2 or a fragment thereof, such as, e.g., amino acids 286-295, 281-300,or 266-315 of SEQ ID NO:2. In other embodiments, the RAF1 fusion proteinis an LMNA:RAF1 fusion protein having the amino acid sequence of SEQ IDNO:4 or a fragment thereof, such as, e.g., amino acids 562-572, 556-575,or 542-591 of SEQ ID NO:4. In some embodiments, the RAF1 fusion proteinis an MPRIP:RAF1 fusion protein having the amino acid sequence of SEQ IDNO:6 or a fragment thereof, such as, e.g., amino acids 1011-1020,1006-1025, or 991-1040 of SEQ ID NO:6. In other embodiments, the RAF1fusion protein is a PAPD7:RAF1 fusion protein having the amino acidsequence of SEQ ID NO:8, or a fragment thereof, such as, e.g., aminoacids 419-428, 414-433, or 399-448 of SEQ ID NO:8. In some embodiments,the RAF1 fusion protein is a CLCN6:RAF1 fusion protein having the aminoacid sequence of SEQ ID NO:10 or a fragment thereof, such as, e.g.,amino acids 45-54, 40-59, or 25-74 of SEQ ID NO:10. In otherembodiments, the RAF1 fusion protein is a TRAK1:RAF1 fusion proteinhaving the amino acid sequence of SEQ ID NO:12 or a fragment thereof,such as, e.g., amino acids 321-330, 316-335, or 301-350 of SEQ ID NO:12.

In some embodiments, the RAF1 fusion protein is an AGGF1:RAF1 fusionprotein comprising an amino acid sequence that is at least 85%, at least90%, at least 95%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO:2 or a fragment thereof (e.g., amino acids 286-295,281-300, or 266-315 of SEQ ID NO:2). In other embodiments, the RAF1fusion protein is an LMNA:RAF1 fusion protein comprising au amino acidsequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO:4 or a fragmentthereof (e.g., amino acids 562-572, 556-575, or 542-591 of SEQ ID NO:4).In yet other embodiments, the RAF1 fusion protein is an MPRIP:RAF1fusion protein comprising an amino acid sequence that is at least 85%,at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO:6 or a fragment thereof (e.g., amino acids1011-1020, 1006-1025, or 991-1040 of SEQ ID NO:6). In some embodimentsthe RAF1 fusion protein is a PAPD7:RAF1 fusion protein comprising anamino acid sequence that is at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 or afragment thereof (e.g., amino acids 419-428, 414-433, or 399-448 of SEQID NO:8). In some embodiments, the RAF1 fusion protein is a CLCN6:RAF1fusion protein comprising an amino acid sequence that is at least 85%,at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO:10 or a fragment thereof (e.g., amino acids45-54, 40-59, or 25-74 of SEQ ID NO:10). In other embodiments, the RAF1fusion protein is a TRAK1:RAF1 fusion protein comprising an amino acidsequence that is at least 85%, at least 90%, at least 95%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO:12 or a fragmentthereof (e.g., amino acids 321-330, 316-335, or 301-350 of SEQ IDNO:12).

In certain embodiments, the RAF1 fusion protein includes a functionalkinase domain. In some embodiments, the RAF1 fusion protein compriseselevated RAF1 activity as compared with wild type RAF1 activity (e.g.,in a cancer cell, a non-cancer cell adjacent to the cancer cell, or anon-cancer cell from a control sample, such as a cancer free subject).In one exemplary embodiment, the RAF1 fusion protein is an AGGF1:RAF1fusion and includes a RAF1 serine/threonine kinase domain or afunctional fragment thereof. In other exemplary embodiments, the RAF1fusion protein is an LMNA:RAF1 fusion and includes a RAF1serine/threonine kinase domain or a functional fragment thereof. In someexemplary embodiments, the RAF1 fusion protein is an MPRIP:RAF1 fusionand includes a RAF1 serine/threonine kinase domain or a functionalfragment thereof. In yet other exemplary embodiments, the RAF1 fusionprotein is a PAPD7:RAF1 fusion and includes a RAF1 serine/threoninekinase domain or a functional fragment thereof. In some exemplaryembodiments, the RAF1 fusion protein is a CLCN6:RAF1 fusion and includesa RAF1 serine/threonine kinase domain or a functional fragment thereof.In other exemplary embodiments, the RAF1 fusion protein is a TRAK1:RAF1fusion and includes a RAF1 serine/threonine kinase domain or afunctional fragment thereof.

In another embodiment, the RAF1 fusion protein or fragment is a peptide,e.g., an immunogenic peptide or protein, that contains a fusion junctionwith a heterologous protein as described herein. Such immunogenicpeptides or proteins can be used for vaccine preparation for use in thetreatment or prevention of cancers caused by or exacerbated by RAF1 genefusions and RAF1 fusion proteins. In other embodiments, such immunogenicpeptides or proteins can be used to raise antibodies specific to thefusion protein. In some embodiments, the RAF1 fusion protein is presentin combination with or is further conjugated to one or more adjuvant(s)or immunogen(s), e.g., a protein capable of enhancing an immune responseto the RAF1 fusion protein (e.g., a hapten, a toxoid, etc.). In someembodiments, the RAF1 fusion protein is an AGGF1:RAF1, LMNA:RAF1,MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1 fusion protein. Insome embodiments, the RAF1 fusion protein comprises the fusion junctionof SEQ ID NO:2, 4, 6, 8, 10, or 12.

Thus, another aspect of the invention provides an antibody that binds toa RAF1 fusion protein (such as, e.g., an AGGF1:RAF1, an LMNA:RAF1, anMPRIP:RAF1, a PAPD7:RAF1, a CLCN6:RAF1, or a TRAK1:RAF1 fusion protein)or a fragment thereof. In certain embodiments, the antibody recognizes aRAF1 fusion protein but does not recognize wild type RAF1 or the wildtype fusion partner (such as, e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, orTRAK1). In some embodiments, the antibody binds to an epitope comprisingthe junction between RAF1 and the fusion partner (e.g., the junction ofAGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, orTRAK1:RAF1). In one embodiment, the antibody binds to an AGGF1:RAF1fusion protein having the amino acid sequence of SEQ ID NO:2 or afragment thereof, such as, e.g., amino acids 286-295, 281-300, or266-315 of SEQ ID NO:2. In other embodiments, the the antibody binds toan LMNA:RAF1 fusion protein having the amino acid sequence of SEQ IDNO:4 or a fragment thereof, such as, e.g., amino acids 562-572, 556-575,or 542-591 of SEQ ID NO:4. In some embodiments, the the antibody bindsto an MPRIP:RAF1 fusion protein having the amino acid sequence of SEQ IDNO:6 or a fragment thereof, such as, e.g., amino acids 1011-1020,1006-1025, or 991-1040 of SEQ ID NO:6. In other embodiments, the theantibody binds to a PAPD7:RAF1 fusion protein having the amino acidsequence of SEQ ID NO:8 or a fragment thereof, such as, e.g., aminoacids 419-428, 414-433, or 399-448 of SEQ ID NO:8. In other embodiments,the the antibody binds to a CLCN6:RAF1 fusion protein having the aminoacid sequence of SEQ ID NO:10 or a fragment thereof, such as, e.g.,amino acids 45-54, 40-59, or 25-74 of SEQ ID NO:10. In yet otherembodiments, the the antibody binds to a TRAK1:RAF1 fusion proteinhaving the amino acid sequence of SEQ ID NO:12 or a fragment thereof,such as, e.g., amino acids 321-330, 316-335, or 301-350 of SEQ ID NO:12.

In certain embodiments, the antibodies of the invention inhibit and/orneutralize the biological activity of the RAF1 fusion protein, and morespecifically, in some embodiments, the kinase activity of the RAF1fusion protein. In other embodiments, the antibodies may be used todetect a RAF1 fusion protein or to diagnose a patient suffering from adisease or disorder associated with the expression of a RAF1 fusionprotein.

Detection and Diagnostic Methods

In another aspect, the invention provides a method of determining thepresence of a RAF1 gene fusion or fusion protein, such as, e.g., anAGGF1:RAF1, an LMNA:RAF1, an MPRIP:RAF1, a PAPD7:RAF1, a CLCN6:RAF1, ora TRAK1:RAF1 fusion as described herein. The presence of a RAF1 fusioncan indicate that the mammal providing the biological sample suffersfrom or is at risk of developing a disorder mediated by aberrant RAF1expression or activity, or overexpression of RAF1, such as, e.g., acancer. The presence of a RAF1 gene fusion may also indicate that thecancer is treatable with a RAF1 inhibitor (such as, e.g., a kinaseinhibitor or an antibody specific to RAF1) or a RAF1 fusion inhibitor.In some embodiments, the RAF1 fusion present in the sample is AGGF1:RAF1and the cancer to be treated is prostate, adenocarcinoma. In otherembodiments, the RAF1 fusion present in the sample is MPRIP:RAF1,LMNA:RAF1, CLCN6:RAF1, or TRAK1:RAF1 and the cancer to be treated ismelanoma. In other embodiments, the cancer is a different cancerassociated with aberrant expression or activity of RAF1 oroverexpression of RAF1.

In one embodiment, the RAF1 fusion detected is a nucleic acid moleculeor a polypeptide. The method includes detecting whether a RAF1 fusionnucleic acid molecule or polypeptide is present in a cell (e.g., acirculating cell or a cancer cell), a tissue (e.g., a tumor), or asample, e.g., a tumor sample, from a subject. In one embodiment, thesample is a nucleic acid sample. In one embodiment, the nucleic acidsample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA. Inother embodiments, the sample is a protein sample.

The sample can be chosen from one or more sample types, such as, forexample, tissue, e.g., cancerous tissue (e.g., a tissue biopsy), wholeblood, serum, plasma, buccal serape, sputum, saliva, cerebrospinalfluid, urine, stool, circulating tumor cells, circulating nucleic acids,or bone marrow.

I. Methods for Detecting Gene Fusions

In some embodiments, the RAF1 fusion is detected in a nucleic acidmolecule by one or more methods chosen from: nucleic acid hybridizationassays (e.g. in situ hybridization, comparative genomic hybridization,microarray, Southern blot, northern blot), amplification-based assays(e.g., PCR, PCR-RFLP assay, or real-time PCR), sequencing and genotyping(e.g. sequence-specific primers, high-performance liquid chromatography,or mass-spectrometric genotyping), and screening analysis (includingmetaphase cytogenetic analysis by karyotype methods).

(1) Hybridization Methods

In some embodiments, the reagent hybridizes to a RAF1 gene fusion, suchas, e.g., nucleotides 868-873, 866-875, 861-880, 846-895, or 821-920 ofSEQ ID NO:1. In alternate embodiments, the reagent detects the presenceof nucleotides 1696-1701, 1693-1702, 1688-1707, 1673-1722, or1648-1747of SEQ NO:3, nucleotides 3043-3048, 3041-3050, 3036-3055,3021-3070, or 2996-3095 of SEQ ID NO:5, nucleotides 1267-1272,1265-1274, 1260-1279, 1245-1294, or 1219-1318 of SEQ ID NO:7,nucleotides 145-150, 143-152, 138-157, 123-172, or 98-197 of SEQ IDNO:9, or nucleotides 973-978, 971-980, 966-975, 951-990, or 926-1015 ofSEQ ID NO:11.

In one embodiment, the method includes: contacting a nucleic acidsample, e.g., a genomic DNA sample (e.g., a chromosomal sample or afractionated, enriched or otherwise pre-treated sample) or a geneproduct (mRNA, or cDNA), obtained from the subject, with a nucleic acidfragment e.g., a probe or primer as described herein (e.g., anexon-specific or a breakpoint-specific probe or primer) under conditionssuitable for hybridization, and determining the presence or absence ofthe RAF1 gene fusion, such as, e.g. AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described herein.

In an alternate embodiment, the method includes the steps of obtaining asample; exposing the sample to a nucleic acid probe which hybridizes toan mRNA or cDNA encoding a RAF1 fusion protein that comprises aminoacids 286-295, 281-300, or 266-315 of SEQ ID NO:2, amino acids 562-572,556-575, or 542-591 of SEQ ID NO:4, amino acids 1011-1020, 1006-1025, or991-1040 of SEQ ID NO:6, amino acids 419-428, 414-433, or 399-448 of SEQID NO:8, amino acids 45-54, 40-59, or 25-74 of SEQ ID NO:10, or aminoacids 321-330, 316-335, or 301-350 of SEQ ID NO:12.

Hybridization, as described throughout the specification, may be carriedout under stringent conditions, e.g., medium or high stringency. See,e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Pr; 2nd edition (1989);T. Brown, Hybridization Analysis of DNA Blots. Current Protocols inMolecular Biology at 21:2.10.1-2.10.16 (2001). High stringencyconditions for hybridization refer to conditions under which two nucleicacids must possess a high degree of base pair homology to each other inorder to hybridize. Examples of highly stringent conditions forhybridization include hybridization in 4×sodium chloride/sodium citrate(SSC), at 65 or 70° C., or hybridization in 4×SSC plus 50% formamide atabout 42 or 50° C., followed by at least one, at least two, or at leastthree washes in 1×SSC, at 65 or 70° C. Another example of highlystringent conditions includes hybridization in 2×SSC; 10×Denhardtsolution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 nMNa₂HPO₄; 250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or 0.25 M ofsodium phosphate buffer, pH 7.2; 1 mM EDTA7% SDS at 60° C.; followed bywashing 2×SSC, 0.1% SDS at 60° C.

The nucleic acid fragments can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label (e.g.,biotin/streptavidin), an antigen lable, or can include an affinity tag,or identifier (e.g., an adaptor, barcode or other sequence identifier).Labeled or unlabeled nucleic acids and/or nucleic acid fragments may beused in reagents for detecting, capturing, or isolating RAF1 genefusions. Labeled or unlabeled nucleic acids and/or nucleic acidfragments may be used in reagents for detecting, capturing, and/orisolating RAF1 gene fusions, such as, e.g., AGGF1:RAF1 (e.g., all orpart of SEQ ID NO: 1), LMNA:RAF1 (e.g., all or part of SEQ ID NO:3),MPRIP:RAF1 (e.g., all or part of SEQ ID NO:5), PAPD7:RAF1 (e.g., all orpall of SEQ ID NO:7), CLCN6:RAF1 (e.g., all or part of SEQ ID NO:9), orTRAK1:RAF1 (e.g., all or part of SEQ ID NO:11). In some embodiments, thelabeled reagent can be detected using, e.g., autoradiography, microscopy(e.g., brightfield, fluorescence, or electron microscopy), enzyme-linkedimmunosorbent assay (ELISA), or immunohistochemistry.

In some embodiments, the method comprises performing chromosome in situhybridization with chromosomal DNA from a biological sample to detectthe presence of a RAF1 gene fusion (such as, e.g., AGGF1:RAF1,LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, asdisclosed herein). In some embodiments, the chromosome in situhybridization comprises the steps of: providing a chromosome (e.g.,interphase or metaphase chromosome) preparation (e.g., by attaching thechromosomes to a substrate (e.g., glass)); denaturing the chromosomalDNA (e.g., by exposure to formamide) to separate the double strands ofthe polynucleotides from each other; exposing the nucleic acid probe tothe chromosomes under conditions to allow hybridization of the probe tothe target DNA; removing unhybridized or non-specifically hybridizedprobes by washing; and detecting the hybridization of the probe with thetarget DNA. In some embodiments, the chromosome in situ hybridization isfluorescence in situ hybridization (FISH). In some embodiments, theprobe is labeled directly by a fluorescent label, or indirectly byincorporation of a nucleotide containing a tag or reporter molecule(e.g., biotin, digoxigenin, or hapten) which after hybridization to thetarget DNA is then bound by fluorescently labeled affinity molecule(e.g., an antibody or streptavidin). In sonic embodiments, thehybridization of the probe with the target DNA in FISH can be visualizedusing a fluorescence microscope.

In other embodiments, the method comprises performing Southern blot withDNA polynucleotides from a biological sample to detect the presence of aRAF1 gene fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein). In someembodiments, the Southern blot comprises the steps of: optionallyfragmenting the polynucleotides into smaller sizes by restrictionendonucleases; separating the polynucleotides by gel electrophoresis;denaturing the polynucleotides (e.g., by heat or alkali treatment) toseparate the double strands of the polynucleotides from each other;transferring the polynucleotides from the gel to a membrane (e.g., anylon or nitrocellulose membrane); immobilizing the polynucleotides tothe membrane (e.g., by UV light or heat); exposing the nucleic acidprobe to the polynucleotides under conditions to allow hybridization ofthe probe to the target DNA; removing unhybridized or non-specificallyhybridized probes by washing; and detecting the hybridization of theprobe with the target DNA.

(2) Amplification-Based Assays

In certain embodiments, the method of determining the presence of a RAF1gene fusion, comprises (a) performing a PCR amplification reaction withpolynucleotides from a biological sample, wherein the amplificationreaction utilizes a pair of primers which will amplify at least afragment of the RAF1 gene fusion, wherein the fragment comprises thefusion junction, wherein the first primer is in sense orientation andthe second primer is in antisense orientation; and (b) detecting anamplification product, wherein the presence of the amplification productis indicative of the presence of a RAF1 fusion polynucleotide in thesample. In specific exemplary embodiments, the RAF1 gene fusion isAGGF1:RAF1, such as, e.g., the gene fusion of SEQ ID NO: 1 or a fragmentthereof comprising nucleotides 868-873, 866-875, 861-880, 846-895, or821-920 of SEQ ID NO:1 In other exemplary embodiments, the gene fissionis LMNA:RAF1 such as, e.g. the gene fusion of SEQ ID NO:3 or a fragmentthereof comprising nucleotides 1696-1701, 1693-1702, 1688-1707,1673-1722, or 1648-1747 of SEQ ID NO:3. In other exemplary embodiments,the gene fusion is MPRIP:RAF1 such as, e.g. the gene fusion of SEQ IDNO:5 or a fragment thereof comprising nucleotides 3043-3048, 3041-3050,3036-3055, 3021-3070, or 2996-3095 of SEQ ID NO:5. In certain exemplaryembodiments, the gene fusion is PAPD7:RAF1 such as, e.g. the gene fusionof SEQ ID NO:7 or a fragment thereof comprising nucleotides 1267-1272,1265-1274, 1260-1279, 1245-1294, or 1219-1318 of SEQ ID NO:7. In someexemplary embodiments, the gene fusion is CLCN6:RAF1 such as, e.g. thegene fusion of SEQ ID NO:9 or a fragment thereof comprising nucleotides145-150, 143-152, 138-157, 123-172, or 98-197 of SEQ ID NO:9. In otherexemplary embodiments, the gene fusion is TRAK1:RAF1 such as, e.g. thegene fusion of SEQ ID NO:11 or a fragment thereof comprising nucleotides973-978, 971-980, 966-975, 951-990, or 926-1015 of SEQ ID NO:11.

In some embodiments, step (a) of performing a PCR amplification reactioncomprises: (i) providing a reaction mixture comprising thepolynucleotides (e.g., DNA or cDNA) from the biological sample, the pairof primers which will amplify at least a fragment of the RAF1 genefusion wherein the first primer is complementary to a sequence on thefirst strand of the polynucleotides and the second printer iscomplementary to a sequence on the second strand of the polynucleotides,a DNA polymerase, and a plurality of free nucleotides comprisingadenine, thymine cytosine, and guanine (dNTPs); (ii) heating thereaction mixture to a first predetermined temperature for a firstpredetermined time to separate the double strands of the polynucleotidesfrom each other; (iii) cooling the reaction mixture to a secondpredetermined temperature for a second predetermined time underconditions to allow the first and second primers to hybridize with theircomplementary sequences on the first and second strands of thepolynucleotides, and to allow the DNA polymerase to extend the primers;and (iv) repeating steps (ii) and (iii) for a predetermined number ofcycles (e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles).

In some embodiments, the polynucleotides from the biological samplecomprise RNA, and the method further comprises performing a RT-PCRamplification reaction with the RNA to synthesize cDNA as the templatefor subsequent or simultaneous PCR reactions. In some embodiments, theRT-PCR amplification reaction comprises providing a reaction mixturecomprising the RNA, a primer which will amplify the RNA (e.g., asequence-specific primer, a random primer, or oligo(dT)s), a reversetranscriptase, and dNTPs, and heating the reaction mixture to a thirdpredetermined temperature for a third predetermined time underconditions to allow the reverse transcriptase to extend the primer.

(3) Sequencing and Genotyping

Another method for determining the presence of a RAF1 gene fusionmolecule (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) includes: sequencing aportion of the nucleic acid molecule (e.g., sequencing the portion ofthe nucleic acid molecule that comprises the fusion junction of a RAF1gene fusion), thereby determining that the RAF1 gene fusion is presentin the nucleic acid molecule. Optionally, the sequence acquired iscompared to a reference sequence, or a wild type reference sequence. Inone embodiment, the sequence is determined by a next generationsequencing method. In some embodiments, the sequencing is automatedand/or high-throughput sequencing. The method can further includeacquiring, e.g., directly or indirectly acquiring, a sample, e.g., atumor or cancer sample, from a patient.

In some embodiments, the sequencing comprises chain terminatorsequencing (Sanger sequencing) comprising: providing a reaction mixturecomprising a nucleic acid molecule from a biological sample, a primercomplementary to a region of the template nucleic acid molecule, a DNApolymerase, a plurality of free nucleotides comprising adenine, thymine,cytosine, and guanine (dNTPs), and at least one chain terminatingnucleotide (e.g., at least one di-deoxynucleotide (ddNTPs) chosen fromddATP, ddTTP, ddCTP, and ddGTP), wherein the at least one chainterminating nucleotide is present in a low concentration so that chaintermination occurs randomly at any one of the positions containing thecorresponding base on the DNA strand; annealing the primer to a singlestrand of the nucleic acid molecule; extending the primer to allowincorporation of the chain terminating nucleotide by the DNA polymeraseto produce a series of DNA fragments that are terminated at positionswhere that particular nucleotide is used; separating the polynucleotidesby electrophoresis (e.g., gel or capillary electrophoresis); anddetermining the nucleotide order of the template nucleic acid moleculebased on the positions of chain termination on the DNA fragments. Insome embodiments, the sequencing is carried out with four separatebase-specific reactions, wherein the primer or the chain terminatingnucleotide in each reaction is labeled with a separate fluorescentlabel. In other embodiments, the sequencing is carried out in a singlereaction, wherein the four chain terminating nucleotides mixed in thesingle reaction are each labeled with a separate fluorescent label.

In some embodiments, the sequencing comprises pyrosequencing (sequencingby synthesis), comprising: (i) providing a reaction mixture comprising,a nucleic acid molecule from a biological sample, a primer complementaryto a region of the template nucleic acid molecule, a DNA polymerase, afirst enzyme capable of converting pyrophosphate into ATP, and a secondenzyme capable using ATP to generates a detectable signal (e.g., achemiluminescent signal, such as light) in an amount that isproportional to the amount of ATP; (ii) annealing the primer to a singlestrand of the nucleic acid molecule; (iii) adding one of the four freenucleotides (dNTPs) to allow incorporation of the correct, complementarydNTP onto the template by the DNA polymerase and release ofpyrophosphate stoichiometrically; (iv) converting the releasedpyrophosphate to ATP by the first enzyme; (v) generating a detectablesignal by the second enzyme using the ATP; (vi) detecting the generatedsignal and analyzing the amount of signal generated in a program; (vii)removing the unincorporated nucleotides; and (viii) repeating steps(iii) to (vii). The method allows sequencing of a single strand of DNA,one base pair at a time, and detecting which base was actually added ateach step. The solutions of each type of nucleotides are sequentiallyadded and removed from the reaction. Light is produced only when thenucleotide solution complements the first unpaired base of die template.The order of solutions which produce detectable signals allows thedetermination of the sequence of the template.

In some embodiments, the method of determining the presence of a RAF1fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) comprises analyzing anucleic acid sample (e.g., DNA, cDNA, or RNA, or an amplificationproduct thereof) by HPLC. The method may comprise: passing a pressurizedliquid solution containing the sample through a column filled with asorbent, wherein the nucleic acid or protein components in the sampleinteract differently with the sorbent, causing different flow rates forthe different components; separating the components as they flow out thecolumn at different flow rates. In some embodiments, the HPLC is chosenfrom, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange HPLC,and bioaffinity HPLC.

In sonic embodiments, the method of determining the presence of a RAF1fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) comprises analyzing anucleic acid sample (e.g., DNA, eDNA, or RNA, or an amplificationproduct thereof) by mass spectrometry. The method may comprise: ionizingthe components in the sample (e.g., by chemical or electron ionization);accelerating and subjecting the ionized components to an electric ormagnetic field; separating the ionized components based on theirmass-to-charge ratios; and detecting the separated components by adetector capable of detecting charged particles (e.g., by an electronmultiplier).

II. Methods for Detecting Fusion Proteins

Another aspect of the invention provides a method of determining thepresence of a RAF1 fusion protein in a mammal. The method comprises thesteps of obtaining a biological sample of a mammal (such as, e.g., ahuman cancer), and exposing that sample to at least one reagent thatdetects a RAF1 fusion protein (e.g., an antibody that recognizes theRAF1 fusion but does not recognize the wild type RAF1 or the wild typefusion partner) to determine whether a RAF1 fusion protein is present inthe biological sample. The detection of a RAF1 fusion protein indicatesthe presence of a mutant RAF1 in the mammal (such as, e.g., in the humancancer). In some embodiments, the RAF1 fusion protein comprises an aminoacid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% identitywith an amino acid sequence of all or part of SEQ ID NO:2, 4, 6, 8, 10,or 12. In some embodiments the cancer is melanoma. In some embodiments,the cancer is prostate adenocarcinoma.

In some embodiments, the reagent that detects a RAF1 fusion protein canbe detectably labeled with, e.g., a radiolabel, a fluorescent label, abioluminescent label, a chemiluminescent label, an enzyme label, abinding pair label (e.g., biotin/streptavidin), an antigen label, or caninclude an affinity tag or identifier (e.g., an adaptor, barcode orother sequence identifier). In some embodiments, the labeled reagent canbe detected using, e.g., autoradiography, microscopy (e.g., brightfield,fluorescence, or electron microscopy), ELISA, or immunohistochemistry.In some embodiments, the RAF1 fusion protein is detected in a biologicalsample by a method chosen from one or more of: antibody-based detection(e.g., western blot, ELISA, immunohistochemistry), size-based detectionmethods (e.g., HPLC or mass spectrometry), or protein sequencing.

(1) Antibody-Based Detection

In some embodiments, the method comprises performing a western blot withpolypeptides from a biological sample to detect the presence of a RAF1fusion protein (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein). In someembodiments, the western blot comprises the steps of: separating thepolypeptides by gel electrophoresis; transferring the polypeptides fromthe gel to a membrane (e.g., a nitrocellulose or polyvinylidenedifluoride (PVDF) membrane); blocking the membrane to preventnonspecific binding by incubating the membrane in a dilute solution ofprotein (e.g., 3-5% bovine serum albumin (BSA) or non-fat dry milk inTris-Buffered Saline (TBS) or I-Block, with a minute percentage (e.g.,0.1%) of detergent, such as, e.g., Tween 20 or Triton X-100); exposingthe polypeptides to at least one reagent that detects a RAF1 fusionprotein (e.g., an antibody that recognizes the RAF1 fusion but does notrecognize the wild type RAF1 or the wild type fusion partner); removingunbound or non-specifically bound reagent by washing; and detecting thebinding of the reagent with the target protein. In some embodiments, themethod comprises two-step detection: exposing the polypeptides to aprimary antibody that specifically binds to a RAF1 fusion protein;removing unbound or non-specifically hound primary antibody by washing;exposing the polypeptides to a secondary antibody that recognizes theprimary antibody; removing unbound or non-specifically bound secondaryantibody by washing; and detecting the binding of the secondaryantibody. In some embodiments, the reagent that detects a RAF1 fusionprotein (e.g., the fusion specific antibody, or the secondary antibody)is directly labeled for detection. In other embodiments, the reagent islinked to an enzyme, and the method further comprises adding a substrateof the enzyme to the membrane; and developing the membrane by detectinga detectable signal produced by the reaction between the enzyme and thesubstrate. For example, the reagent may be linked with horseradishperoxidase to cleave a chemiluminescent agent as a substrate, producingluminescence in proportion to the amount of the target protein fordetection.

In some embodiments, the method comprises performing ELISA withpolypeptides from a biological sample to detect the presence of a RAF1fusion protein (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein). In someembodiments, the ELISA is chosen from, e.g., direct ELISA, indirectELISA, sandwich ELISA, and competitive ELISA.

In one embodiment, the direct ELISA comprises the steps of: attachingpolypeptides from a biological sample to a surface; blocking the surfaceto prevent nonspecific binding by incubating the surface in a dilutesolution of protein; exposing the polypeptides to an antibody thatspecifically binds to a RAF1 fusion protein (e.g., an antibody thatrecognizes the RAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1,MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein)but does not recognize the wild type RAF1 or the wild type fusionpartner); removing unbound or non-specifically bound antibody bywashing; and detecting the binding of the antibody with the targetprotein. In some embodiments, the antibody is directly labeled fordetection. In other embodiments, the antibody is linked to an enzyme,and the method further comprises adding a substrate of the enzyme; anddetecting a detectable signal produced by the reaction between theenzyme and the substrate.

In another embodiment, the indirect ELISA comprises the steps of:attaching polypeptides from a biological sample to a surface; blockingthe surface to prevent nonspecific binding by incubating the surface ina dilute solution of protein; exposing the polypeptides to a primaryantibody that specifically binds to a RAF1 fusion protein (such as,e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, orTRAK1:RAF1, as disclosed herein); removing unbound or non-specificallybound primary antibody by washing; exposing the polypeptides to asecondary antibody that recognizes the primary antibody; removingunbound or non-specifically bound secondary antibody by washing; anddetecting the binding of the secondary antibody. In some embodiments,the secondary antibody is directly labeled for detection. In otherembodiments, the secondary antibody is linked to an enzyme, and themethod further comprises adding a substrate of the enzyme; and detectinga detectable signal produced by the reaction between the enzyme and thesubstrate.

In some embodiments, the method comprises performingimmunohistochemistry with polypeptides from a biological sample todetect the presence of a RAF1 fusion protein (such as, e.g., AGGF1:RAF1,LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, asdisclosed herein). In some embodiments, the immunohistochemistrycomprises the steps of: fixing a cell or a tissue section (e.g., byparaformaldehyde or formalin treatment); permeabilizing the cell ortissue section to allow target accessibility; blocking the cell ortissue section to prevent nonspecific binding; exposing the cell ortissue section to at least one reagent that detects a RAF1 fusionprotein (e.g., an antibody that recognizes the RAF1 fusion but does notrecognize the wild type RAF1 or the wild type fusion partner); removingunbound or non-specifically bound reagent by washing; and detecting thebinding of the reagent with the target protein. In some embodiments, thereagent is directly labeled for detection. In other embodiments, thereagent is linked to an enzyme, and the method further comprises addinga substrate of the enzyme; and detecting a detectable signal produced bythe reaction between the enzyme and the substrate. In some embodiments,the immunohistochemistry may comprise the two-step detection as in theindirect ELISA.

(2) Size-Based Detection Methods

In some embodiments, the method of determining the presence of a RAF1fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) comprises analyzing aprotein sample by HPLC. The method may comprise: passing a pressurizedliquid solution containing the sample through a column tilled with asorbent, wherein the nucleic acid or protein components in the sampleinteract differently with the sorbent, causing different flow rates forthe different components; separating the components as they flow out thecolumn at different flow rates. In some embodiments, the HPLC is chosenfrom, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange HPLC,and bioaffinity HPLC.

In some embodiments, the method of determining the presence of a RAF1fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as disclosed herein) comprises analyzing aprotein sample by mass spectrometry. The method may comprise: ionizingthe components in the sample (e.g., by chemical or electron ionization);accelerating and subjecting the ionized components to an electric ormagnetic field; separating the ionized components based on theirmass-to-charge ratios; and detecting the separated components by adetector capable of detecting charged particles (e.g., by an electronmultiplier).

Detection of a RAF1 gene fusion or a RAF1 fusion protein in a patientcan lead to assignment of the patient to the newly identified patientpopulation that bears the RAF1 fusion. Because this patient populationcan suffer from or be susceptible to a disorder associated with aberrantRAF1 expression or activity or overexpression of RAF1, detection of theRAF1 fusion can also lead to diagnosis of such disorder. Thus, a furtheraspect of the invention provides a method of stratifying a patientpopulation (e.g., assigning a patient, to a group or class) and/ordiagnosing a patient, comprising: obtaining a biological sample from thepatient, contacting the sample with at least one reagent that detects aRAF1 gene fusion or a RAF1 fusion protein to determine whether a RAF1fusion is present in the biological sample. The detection of a RAF1fusion indicates that the patient belongs to the newly identifiedpatient population that bears the RAF1 fusion, and/or the presence of adisorder associated with aberrant RAF1 expression or activity oroverexpression of RAF1, such as e.g., a cancer. The detection of a RAF1fusion also identities a new subtype of cancer, which is characterizedby the presence of the RAF1 fusion. In some embodiments, the cancer ismelanoma. In sonic embodiments, the cancer is prostate adenocarcinoma.In certain embodiments, the RAF1 fusion is AGGF1:RAF1. In otherembodiments, the RAF1 fusion is LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1. In sonic embodiments, the AGGF1:RAF1 fusionhas all or a part of the nucleotide and/or amino acid sequence (such as,e.g., the fusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:2,respectively. In some embodiments, the LMNA:RAF1 fusion has all or partof the nucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ NO:3 and SEQ ID NO:4, respectively. In someembodiments, the MPRIP:RAF1 fusion has all or part of the nucleotideand/or amino acid sequence (such as, e.g., the fusion junction) setforth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments,the PAPD7:RAF1 fusion has all or part of the nucleotide and/or aminoacid sequence (such as, e.g., the fusion junction) set forth in SEQ IDNO:7 and SEQ ID NO:8, respectively. In some embodiments, the CLCN6:RAF1fusion has all or part of the nucleotide and/or amino acid sequence(such as, e.g., the fusion junction) set forth in SEQ ID NO:9 and SEQ IDNO:10, respectively. In some embodiments, the TRAK1:RAF1 fusion has allor part of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:11 and SEQ ID NO:12,respectively.

In some embodiments, the RAF1 gene fusion or RAF1 fusion protein isdetected prior to initiating, during, and/or after, a treatment of apatient with, e.g., a RAF1 inhibitor or a RAF1 fusion inhibitor. In oneembodiment, the RAF1 gene fusion or RAF1 fusion protein is detected atthe time the patient is diagnosed with a cancer. In other embodiment,the RAF1 fusion is detected at a pre-determined interval, e.g., a firstpoint in time and at least at a subsequent point in time. In certainembodiments, in response to detection of a RAF1 fusion, such as, e.g.,AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, orTRAK1:RAF1, the method further includes one or more of:

(1) stratifying a patient population (e.g., assigning a patient, to agroup or class);

(2) identifying or selecting the patient as likely or unlikely torespond to a treatment, e.g., a RAF1 inhibitor treatment (e.g., a kinaseinhibitor treatment), or a RAF1 fusion inhibitor treatment as describedherein;

(3) selecting a treatment regimen, e.g., administering or notadministering a preselected therapeutic agent, such as, e.g., a RAF1inhibitor, or a RAF1 fusion inhibitor;

(4) prognosticating the time course of the disease in the patient e.g.,evaluating the likelihood of increased or decreased patient survival);or

(5) monitoring the effectiveness of treatment (e.g., by detecting areduction in the level of RAF1 gene fusion or fusion protein in apatient sample).

In certain embodiments, upon detection of a RAF1 gene fusion or RAF1fusion protein in a patient's biological sample, the patient isidentified as likely to respond to a treatment that comprises a RAF1inhibitor, or a RAF1 fusion inhibitor. In some embodiments, the RAF1fusion detected is an AGGF1:RAF1 fusion. In alternate embodiments, theRAF1 fusion detected is an MPRIP:RAF1 fusion. In some embodiments, theRAF1 fusion detected is a PAPD7:RAF1 fusion. In some embodiments, theRAF1 fusion detected is a LMNA:RAF1 fusion. In some embodiments, theRAF1 fusion detected is a CLCN6:RAF1 fusion. In sonic embodiments, theRAF1 fusion detected is a TRAK1:RAF1 fusion.

A further aspect of the invention provides a method of selecting atreatment option by detecting a RAF1 fusion. The method comprisesobtaining a biological sample from a patient and exposing the sample toat least one reagent that detects a RAF1 gene fusion or fusion proteinto determine whether a RAF1 fusion is present in the biological sample.The detection of the RAF1 fusion indicates the likelihood of the patientresponding to treatment with a RAF1 inhibitor, or a RAF1 fusioninhibitor. The method may be augmented or personalized by evaluating theeffect of a variety of kinase, RAF1 or RAF1 fusion inhibitors on thebiological sample shown to contain a RAF1 fusion to determine the mostappropriate inhibitor to administer. In certain embodiments, the RAF1fusion is AGGF1:RAF1. In other embodiments, the RAF1 fusion isLMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1. In someembodiments, the AGGF1:RAF1 fusion has all or a part of the nucleotideand/or amino acid sequence (such as, e.g., the fusion junction) setforth in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments,the LMNA:RAF1 fusion has all or part of the nucleotide and/or amino acidsequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:3and SEQ ID NO:4, respectively. In some embodiments, the MPRIP:RAF1fusion has all or part of the nucleotide and/or amino acid sequence(such as, e.g., the fusion junction) set forth in SEQ ID NO:5 and SEQ IDNO:6, respectively. In some embodiments, the PAPD7:RAF1 fusion has allor part of the nucleotide and/or amino acid sequence (such as, e.g., thefusion junction) set forth in SEQ ID NO:7 and SEQ NO:8, respectively. Insome embodiments, the CLCN6:RAF1 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:10 and SEQ ID NO:11, respectively. Insome embodiments, the TRAK1:RAF1 fusion has all or part of thenucleotide and/or amino acid sequence (such as, e.g., the fusionjunction) set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively.

Methods of Treatment

Alternatively, or in combination with the detection and diagnosticmethods described herein, the invention provides method for treating thenewly identified patient population and the new RAF1 fusion cancersubtype, which are characterized by the presence of a RAF1 fusion. Thepatient population and cancer subtype can be associated with or predictthe onset of a condition mediated by aberrant RAF1 expression oractivity, or overexpression of RAF1, such as, e.g., a cancer or a tumorharboring a RAF1 fusion. The methods comprise administering atherapeutic agent, e.g., a RAF1 inhibitor, such as e.g., a kinaseinhibitor or an antibody specific to RAF1; or a RAF1 fusion inhibitor,i.e., an inhibitor that blocks the activity of the RAF1 fusion but notwild type RAF1 or wild type fusion partner, such as e.g., an antibodyspecific to an AGGF1:RAF1, an LMNA:RAF1, an MPRIP:RAF1, a PAPD7:RAF1, aCLCN6:RAF1, or a TRAK1:RAF1 fusion protein, e.g., any one of theantibodies described above, or an RNA inhibitor that recognizes RAF1 orthe fusion junction of a RAF1 gene fusion, including but not limited tosiRNA, dsRNA, shRNA, or any other antisense nucleic acid inhibitor,alone or in combination with e.g., other chemotherapeutic agents orprocedures, in an amount sufficient to treat a condition mediated byaberrant RAF1 expression Of activity, or overexpression of RAF1 by oneor more of the following: impeding growth of a cancer, causing a cancerto shrink by weight or volume, extending the expected survival time ofthe patient, inhibiting tumor growth, reducing tumor mass, reducing sizeor number of metastatic lesions, inhibiting the development of newmetastatic lesions, prolonging survival, prolonging progression-freesurvival, prolonging time to progression, and/or enhancing quality oflife.

In certain embodiments, the RAF1 fusion of the invention may beinhibited by a RAF1 inhibitor or a RAF1 fusion inhibitor. In someembodiments, the therapeutic agent is a RAF1 inhibitor, such as, e.g., acompound, biological or chemical, which inhibits, directly orindirectly, the expression and/or activity of RAF1. For example, theRAF1 inhibitors may be an antibody (such as, e.g., antibodies specificto RAF1) or a small molecule inhibitor (such as, e.g., a kinaseinhibitor). In some embodiments, the inhibitors may act directly on RAF1itself, modify the activity of RAF1, or inhibit the expression of RAF1.In other embodiments, the inhibitors may indirectly inhibit RAH activityby inhibiting the activity of proteins or molecules other than RAF1itself. For example, the inhibitors may modulate the activity ofregulatory kinases that phosphorylate or dephosphorylate RAF1, interferewith binding of ligands, or inhibit the activity of interacting ordownstream proteins or molecules.

In some embodiments, the RAF1 fusion is inhibited by a RAF1 fusioninhibitor, such as, e.g., an antibody that recognizes all or part of aRAF1 fusion (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described herein) but does notrecognize wild type RAF1 or wild type fusion partner (e.g., AGGF1, LMNA,MPRIP, PAPD7, CLCN6, or TRAK1). In some embodiments, the RAF1 fusionprotein (such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1,CLCN6:RAF1, or TRAK1:RAF1, as described herein) is inhibited by an agentthat inhibits transcription or translation of the fusion protein, e.g.,an RNA inhibitor that recognizes the RAF1 coding sequence, the bindingpartner (e.g., AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1), or thebinding partner: RAF1 fusion junction, including but not limited tosmall interfering RNA (siRNA), double stranded RNA (dsRNA),short-hairpin RNA (shRNA), or any other antisense nucleic acidinhibitor. In some embodiments, the RAF1 fusion inhibited is selectedfrom all or a portion of any one of SEQ ID NOs: 1-12.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of a conditionmediated by aberrant RAF1 expression or activity, or overexpression ofRAF1, such as, delaying or minimizing one or more symptoms associatedwith a cancer or a tumor harboring a RAF1 fusion (such as, e.g.,AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, orTRAK1:RAF1, as described herein). A therapeutically effective amount ofa compound means an amount of therapeutic agent, alone or in combinationwith other therapeutic agents, which provides a therapeutic benefit inthe treatment or management of the cancer. The term “therapeuticallyeffective amount” can encompass an amount that improves overall therapy,reduces or avoids symptoms or causes of the condition mediated byaberrant RAF1 expression activity or overexpression of RAF1, or enhancesthe therapeutic efficacy of another therapeutic agent.

In certain embodiments, the cancer or tumor harboring a RAF1 fusion ismelanoma. In some embodiments the cancer or tumor harboring a RAF1fusion is a prostate adenocarcinoma.

In some embodiments, the patient to be treated is suffering frommelanoma, and the method for treating the condition comprisesadministering to the patient a therapeutically effective amount of aRAF1 inhibitor or a RAF1 fusion inhibitor as described above. In someembodiments, the patient to be treated is suffering from prostateadenocarcinoma, and the method for treating the condition comprisesadministering to the patient a therapeutically effective amount of aRAF1 inhibitor or a RAF1 fusion inhibitor as described above.

Screening Methods

Therapeutic agents, such as, e.g., RAF1 inhibitors, and RAF1 fusioninhibitors (gene fusion and fusion protein), used in the therapeuticmethods of the invention can be evaluated using the screening assaysdescribed herein. Thus, the invention provides a method of identifyingan agent useful for treating a condition mediated by aberrant RAF1expression or activity, or overexpression of RAF1, e.g., cancer or atumor harboring a RAF1 fusion, such as, e.g., melanoma or prostateadenocarcinoma, comprising contacting a cell expressing a RAF1 genefusion or RAF1 fusion protein with a candidate agent and using one ofthe detection methods referenced above to determine whether theexpression level of the fusion is decreased or a biological functionassociated with the fusion is altered. In one embodiment, therapeuticagents can be evaluated in a cell-free system, e.g., a cell lysate or ina reconstituted system. In other embodiments, the therapeutic agents areevaluated in a cell in culture, e.g., a cell expressing a RAF1 fusion(e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell).In yet other embodiments, the therapeutic agents are evaluated in a cellin vivo (a RAF1 fusion-expressing cell present in a subject, e.g., ananimal subject (e.g., an in vivo animal model).

Exemplary parameters to evaluate in determining the efficacy of atherapeutic agent for treating a condition mediated by aberrant RAF1expression or activity, or overexpression of RAF1, such as, e.g., acancer or a tumor harboring a RAF1 fusion include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a RAF1 fusion protein or a binding competition between a knownligand and the candidate agent to a RAF1 fusion protein;

(ii) a change in kinase activity, e.g., phosphorylation levels of a RAF1fusion protein (e.g., an increased or decreased phosphorylation orautophosphorylation) or a change in phosphorylation of a target of aRAF1 kinase—in certain embodiments, a change in kinase activity, e.g.,phosphorylation, is detected by any of western blot (e.g., using ananti-RAF1 antibody or a phosphor-specific antibody, detecting a shift inthe molecular weight of a RAF1 fusion protein), mass spectrometry,immunoprecipitation, immunohistochemistry, immunomagnetic beads, amongothers;

(iii) a change in an activity of a cell containing a RAF1 fusion (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology, or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, or proliferation of the tumor;

(v) a change in the level, e.g., expression level, of a RAF1 fusionprotein or nucleic acid molecule; or

(vi) a change in an activity of a signaling pathway involving RAF1,e.g., phosphorylation or activity of an interacting or downstreamtarget, or expression level of a target gene.

In some embodiments, the RAF1 fusion is an AGGF1:RAF1 fusion, anLMNA:RAF1 fusion, an MPRIP:RAF1 fusion, a PAPD7:RAF1 fusion, aCLCN6:RAF1 fusion, or a TRAK1:RAF1 fusion.

In one embodiment, a change in the activity of a RAF1 fusion, orinteraction of a RAF1 fusion with a downstream ligand detected in a cellfree assay in the presence of a candidate agent indicates that thecandidate agent will be effective as a therapeutic agent for treatmentof a condition mediated by aberrant RAF1 expression or activity, oroverexpression of RAF1, such as, e.g., a cancer or a tumor harboring aRAF1 fusion. In some embodiments, the cancer or tumor is postateadenocarinoma. In some embodiments, the cancer or tumor is melanoma.

In other embodiments, a change in an activity of a cell expressing aRAF1 fusion, such as, e.g., AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1,PAPD7:RAF1, CLCN6:RAF1, or TRAK1:RAF1, as described herein, (e.g., amammalian cell, a tumor cell or cell line, a recombinant cell) isdetected in a cell in culture. In one embodiment, the cell is arecombinant cell that is modified to express a RAF1 fusion nucleic acid,e.g., is a recombinant cell transfected with a RAF1 fusion nucleic acid.The transfected cell can show a change in response to the expressed RAF1fusion, e.g., increased proliferation, changes in morphology, increasedtumorigenicity, and/or acquired a transformed phenotype. A change in anyof the activities of the cell, e.g., the recombinant cell, in thepresence of the candidate agent can be detected. For example, a decreasein one or more of: proliferation, tumorigenicity, or transformedmorphology, in the presence of the candidate agent can be indicative ofan inhibitor of a RAF1 fusion. In other embodiments, a change in bindingactivity or phosphorylation of RAF1 or its interacting or downstreamproteins or molecules as described herein is detected.

In yet other embodiment, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,a tumor containing animal or a xenograft comprising cells expressing aRAF1 fusion (e.g., tumorigenic cells expressing a RAF1 fusion) isemployed. The therapeutic agents can be administered to the animalsubject and a change in the tumor is evaluated. In one embodiment, thechange in the tumor includes one Of more of a tumor growth, tumor size,tumor burden, or survival is evaluated. A decrease in one or more oftumor growth, tumor size, tumor burden, or an increased survival isindicative that the candidate agent is an inhibitor or modulator.

In another aspect of the invention provides a method or assay forscreening for agents that modulate (e.g., inhibit) the expression oractivity of a RAF1 fusion as described herein. The method includescontacting e.g., a RAF1 fusion, or a cell expressing a RAF1 fusion, witha candidate agent; and detecting a change in a parameter associated witha RAF1 fusion, e.g., a change in the expression or an activity of theRAF1 fusion. The method can, optionally, include comparing the treatedparameter to a reference value, e.g., a control sample (e.g., comparinga parameter obtained from a sample with the candidate agent to aparameter obtained from a sample without the candidate agent). In oneembodiment, if a decrease in expression or activity of the RAF1 fusionis detected, the candidate agent is identified as an inhibitor. Inanother embodiment, if an increase in expression or activity of the RAF1fusion is detected, the candidate agent is identified as an activator.In certain embodiments, the RAF1 fusion is a RAF1 gene fusion or RAF1fusion protein, such as, e.g., an AGGF1:RAF1 fusion. an MPRIP:RAF1fusion, a PAPD7:RAF1 fusion, an LMNA:RAF1 fusion, a CLCN6:RAF1 fusion,or a TRAK1:RAF1 fusion.

In one embodiment, the contacting step is detected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is detected in a cell in culture, e.g.,a cell expressing a RAF1 fusion (e.g., a mammalian cell, a tumor cell orcell line, a recombinant cell). In yet other embodiments, the contactingstep is detected in a cell in vivo (e.g., a RAF1 expressing cell presentin a subject, e.g., an animal subject (e.g., an in vivo animal model)).

Exemplary parameters evaluated in identifying an agent that modulatesthe activity of a RAF1 fusion (e.g., an AGGF1:RAF1 fusion, an MPRIP:RAF1fusion, a PAPD7:RAF1 fusion, an LMNA:RAF1 fusion, a CLCN6:RAF1 fusion,or a TRAK1:RAF1 fusion) include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a RAF1 fusion protein; a binding competition between a knownligand and the candidate agent to a RAF1 fusion protein;

(ii) a change in kinase activity, e.g., phosphorylation levels of a RAFi fusion protein (e.g., an increased or decreased phosphorylation orautophosphorylation) or a change in phosphorylation of a target of aRAF1 kinase—in certain embodiments, a change in kinase activity, e.g.,phosphorylation, is detected by any of Western blot (e.g., using ananti-RAF1 antibody or a phosphor-specific antibody, detecting a shift inthe molecular weight of a RAF1 fusion protein), mass spectrometry,immunoprecipitation, immunohistochemistry, immunomagnetic beads, amongothers;

(iii) a change in an activity of a cell containing a RAF1 fusion (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology, or tumorigenicity of the cell;

(iv) a change in tumor present in an annual subject, e.g., size,appearance, or proliferation of the tumor;

(v) a change in the level, e.g., expression level, of a RAF1 fusionprotein or nucleic acid molecule; or

(vi) a change in an activity of a signaling pathway involving RAF1,e.g., phosphorylation or activity of an interacting or downstreamtarget, or expression level of a target gene.

Methods for Validating RAF1 Fusions

RAF1 gene fusions (e.g., AGGF1:RAF1 gene fusions, MPRIP:RAF1 genefusions, PAPD7:RAF1 gene fusions, LMNA:RAF1 gene fusions, CLCN6:RAF1gene fusions, or TRAK1:RAF1 gene fusions) may be evaluated to ensurethat the breakpoints are in-frame and can produce a protein productcontaining the full kinase domain, i.e., that the breakpoint occurs suchthat complete triplet codons are intact, and that the RNA sequence willproduce a viable protein. The RAF1 gene fusion can be transfected intocells to confirm that the protein is functionally active with respect tokinase activity and oncogenic activity. cDNA encoding the RAF1 fusionprotein can be produced by standard solid-phase DNA synthesis.Alternatively the RAF1 fusion cDNA can be produced by RT-PCR using tumormRNA extracted from samples containing the gene fusion. The DNAamplified can be subcloned into an appropriate vector and characterizedby DNA sequence analysis or in vitro/in vivo expression analyses.

Expression vectors containing the RAF1 gene fusion (such as, e.g., aRAF1 gene fusion, e.g., an AGGF1:RAF1 gene fusion, an MPRIP:RAF1 genefusion, a PAPD7:RAF1 gene fusion, an LMNA:RAF1 gene fusion, a CLCN6:RAF1gene fusion, or a TRAK1:RAF1 gene fusion) can be introduced into hostcells to thereby produce a RAF1 fusion protein (such as, e.g., a RAF1fusion protein, e.g., an AGGF1:RAF1 fusion protein, an MPRIP:RAF1 fusionprotein, a PAPD7:RAF1 fusion protein, an LMNA:RAF1 fusion protein, aCLCN6:RAF1 fusion protein, or a TRAK1:RAF1 fusion protein). The RAF1fusion protein expression vector can be a yeast expression vector, avector for expression in insect cells, e.g., a baculovirus expressionvector, or a vector suitable for expression in mammalian cells. VectorDNA can be introduced into host cells via conventional transformation ortransfection techniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell.

Cells harboring the expression vector carrying the recombinant RAF1 genefusion can then be tested for production of the unique fusion proteinvia standard Western blotting using either an antibody probe thatdetects the gene product itself or that recognizes a tag peptide (e.g.,FLAG tag) that can be added to the gene product via the expressionvector (using standard, commercially available reagents). Westernblotting can be used to confirm the ectopic expression of the encodedRAF1 fusion protein by comparing the samples from cells transfected withthe vector containing the RAF1 gene fusion cDNA to cells transfectedwith the empty expression vector. The functional activity can beassessed by measuring the level of phosphorylation on the kinase orsubstrate. Comparison of the level of phosphorylation activity betweenthe wild type (normal) form of RAF1 and the RAF1 fusion protein canindicate if the RAF1 fusion protein has elevated activity that coulddrive oncogenic activity. Whether the RAF1 gene fusion is oncogenic canbe assessed by measuring capacity of the expressed RAF1 fusion proteinto transform cells, that is, to enable cells to grow and proliferateunder conditions which are not permissive for growth of normal cells.One commonly used method of measuring the transforming activity of akinase is by assessing if expression of the gene product can allow BaF3cells to grow in the absence of the growth factor IL3, which is requiredfor the survival and growth of BaF3 cells. Another assay for measuringtransforming activity is a soft agar growth assay. This is anotherstandard method which tests the capacity of an introduce gene product toconfer the ability to grow in a soft agar matrix, oranchorage-independent conditions. These methods and others can be usedto test the oncogenic activity of a RAF1 gene fusion (such as, e.g., anAGGF1:RAF1 gene fusion, an MPRIP:RAF1 gene fusion, an LMNA:RAF1 genefusion, a PAPD7 gene fusion, a CLCN6:RAF1 gene fusion, or a. TRAK1:RAF1gene fusion) and provide a level of validation of a RAF1 fusion protein(such as, e.g., an AGGF1:RAF1 fusion protein, an MPRIP:RAF1 fusionprotein, a PAPD7:RAF1 fusion protein, an LMNA:RAF1 fusion protein, aCLCN6:RAF1 fusion protein, or a TRAK1:RAF1 fusion protein) as apotential target for treating patients that harbor these fusions.

A change in an activity of a cell can be detected in a cell in culture,e.g., a cell expressing a fusion (e.g., a mammalian cell, a tumor cellor cell line, a recombinant cell). The transfected cell can show achange in response to the expressed fusion, e.g., increasedproliferation, changes in morphology, increased tumorigenicity, and/oran acquired transformed phenotype.

To further validate the biological implication of the gene fusion, achange in any of the activities of the cell, e.g., the recombinant cell,in the presence of a known inhibitor of one of the fusion partners,e.g., a RAF1 inhibitor, can be detected. For example, a decrease in oneor more of: proliferation, tumorigenicity, and transformed morphology,in the presence of the BETA inhibitor can be indicative of an inhibitorof a fusion. In other embodiments, a change in binding activity orphosphorylation of RAF1 or its interacting or downstream proteins ormolecules is detected.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification will supersede any contradictorymaterial. Unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. All ranges given in the application encompass theendpoints unless stated otherwise.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-76. (canceled)
 77. A method for detecting in a patient a RAF1 fusionprotein or a RAF1 gene fusion that results in aberrant activity orexpression of RAF1 or overexpression of RAF1, wherein the RAF1 fusionprotein is an AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1,or a TRAK1:RAF1 fusion protein, and the RAF1 gene fusion is anLMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, or a TRAK1:RAF1 genefusion, said method comprising: a) contacting a biological sample fromthe patient with a reagent selected from i) an antibody thatspecifically binds to the RAF1 fusion protein, but does not specificallybind to wild-type RAF1, AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1; orii) an oligonucleotide that hybridizes to the fusion junction of theRAF1 gene fusion; and b) detecting binding between the RAF1 fusionprotein or the RAF1 gene fusion and the reagent.
 78. The method of claim77, wherein: a) the RAF1 gene fusion comprises: i) SEQ ID NO:3, 5, 7, 9,or 11, or ii) a portion of SEQ ID NO:3, 5, 7, 9, or 11, wherein theportion comprises a fusion junction between RAF1 and its fusion partnerand the gene fusion encodes a polypeptide having RAF1 kinase activity,or b) the RAF1 fusion protein comprises: i) SEQ ID NO:2, 4, 6, 8, 10, or12, or ii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein theportion comprises a fusion junction between RAF1 and its fusion partnerand the fusion protein has RAF1 kinase activity.
 79. The method of claim77, wherein the reagent is an oligonucleotide that hybridizes understringent conditions to: a) a fragment of SEQ ID NO:3 comprisingnucleotides 1693-1702 of SEQ ID NO:3; b) a fragment of SEQ ID NO:5comprising nucleotides 3041-3050 of SEQ ID NO:5; c) a fragment of SEQ IDNO:7 comprising nucleotides 1265-1274 of SEQ ID NO:7; d) a fragment ofSEQ ID NO:9 comprising nucleotides 143-152 of SEQ ID NO:9; e) a fragmentof SEQ ID NO:11 comprising nucleotides 971-980 of SEQ ID NO:11; or f) acomplementary oligonucleotide of any one of a)-e).
 80. The method ofclaim 77, wherein the reagent is an antibody that specifically binds to:a) a fragment of SEQ ID NO:2 comprising amino acids 286-295 of SEQ IDNO:2; b) a fragment of SEQ ID NO:4 comprising amino acids 562-572 of SEQID NO:4; c) a fragment of SEQ ID NO:6 comprising amino acids 1011-1020of SEQ ID NO:6; or d) a fragment of SEQ ID NO:8 comprising amino acids419-428 of SEQ ID NO:8; e) a fragment of SEQ ID NO:10 comprising aminoacids 45-54 of SEQ ID NO:10; or f) a fragment of SEQ ID NO:12 comprisingamino acids 321-330 of SEQ ID NO:12.
 81. The method of claim 77, whereinthe patient is suffering from or susceptible to a cancer.
 82. The methodof claim 81, wherein the cancer is prostate adenocarcinoma or melanoma.83. A method of treating a patient in which a RAF1 fusion proteinselected from AGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1,and TRAK1:RAF1 or a RAF1 gene fusion selected from LMNA:RAF1,MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, and TRAK1:RAF1 has been detected,said method comprising administering to the patient a therapeuticallyeffective amount of a compound that inhibits RAF1 activity or theactivity or expression of the RAF1 fusion protein or RAF1 gene fusion,wherein the RAF1 fusion protein or RAF1 gene fusion has been detected inthe patient by a method comprising: a) contacting a biological samplefrom the patient with a reagent selected from: i) an antibody thatspecifically binds to a RAF1 fusion protein, but does not specificallybind to wild-type RAF1, AGGF1, LMNA, MPRIP, PAPD7, CLCN6, or TRAK1; orii) an oligonucleotide that hybridizes to the fusion junction of a RAF1gene fusion; and b) detecting binding between the RAF1 fusion protein orthe RAF1 gene fusion and the reagent.
 84. The method of claim 83,wherein: a) the RAF1 gene fusion comprises: i) SEQ ID NO:3, 5, 7, 9, or11, or ii) a portion of SEQ ID NO:3, 5, 7, 9, or 11, wherein the portioncomprises a fusion junction between RAF1 and its fusion partner and thegene fusion encodes a polypeptide having RAF1 kinase activity, or b) theRAF1 fusion protein comprises: i) SEQ ID NO:2, 4, 6, 8, 10, or 12, orii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein the portioncomprises a fusion junction between RAF1 and its fusion partner and thefusion protein has RAF1 kinase activity.
 85. The method of claim 83,wherein the reagent is an oligonucleotide that hybridizes understringent conditions to: a) a fragment of SEQ ID NO:3 comprisingnucleotides 1693-1702 of SEQ ID NO:3; b) a fragment of SEQ ID NO:5comprising nucleotides 3041-3050 of SEQ ID NO:5; c) a fragment of SEQ IDNO:7 comprising nucleotides 1265-1274 of SEQ ID NO:7; d) a fragment ofSEQ ID NO:9 comprising nucleotides 143-152 of SEQ ID NO:9; e) a fragmentof SEQ ID NO:11 comprising nucleotides 971-980 of SEQ ID NO:11; or f) acomplementary oligonucleotide of any one of a)-e).
 86. The method ofclaim 83, wherein the reagent is an antibody that specifically binds to:a) a fragment of SEQ ID NO:2 comprising amino acids 286-295 of SEQ IDNO:2; b) a fragment of SEQ ID NO:4 comprising amino acids 562-572 of SEQID NO:4; c) a fragment of SEQ ID NO:6 comprising amino acids 1011-1020of SEQ ID NO:6; or d) a fragment of SEQ ID NO:8 comprising amino acids419-428 of SEQ ID NO:8; e) a fragment of SEQ ID NO:10 comprising aminoacids 45-54 of SEQ ID NO:10; or f) a fragment of SEQ ID NO:12 comprisingamino acids 321-330 of SEQ ID NO:12.
 87. The method of claim 83, whereinthe patient is suffering from or susceptible to a cancer.
 88. The methodof claim 87, wherein the cancer is prostate adenocarcinoma or melanoma.89. A molecule capable of specifically binding to a RAF1 fusion proteinor a RAF1 gene fusion that results in aberrant activity or expression ofRAF1 or overexpression RAF1, wherein the molecule is selected from: a)an antibody that specifically binds to the RAF1 fusion protein selectedfrom AGGF1:RAF1, LMNA: RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, andTRAK1:RAF1, but does not specifically bind to wild-type RAF1, AGGF1,LMNA, MPRIP, PAPD7, CLCN6, or TRAK1; or b) an oligonucleotide thathybridizes to the fusion junction of the RAF1 gene fusion selected fromAGGF1:RAF1, LMNA:RAF1, MPRIP:RAF1, PAPD7:RAF1, CLCN6:RAF1, andTRAK1:RAF1.
 90. The molecule of claim 89, wherein: a) the RAF1 genefusion that the molecule specifically binds to comprises: i) SEQ IDNO:1, 3, 5, 7, 9, or 11, or ii) a portion of SEQ ID NO:1, 3, 5, 7, 9, or11, wherein the portion comprises a fusion junction between RAF1 and itsfusion partner and the gene fusion encodes a polypeptide having RAF1kinase activity, or b) the RAF1 fusion protein that the moleculespecifically binds to comprises: i) SEQ ID NO:2, 4, 6, 8, 10, or 12, orii) a portion of SEQ ID NO:2, 4, 6, 8, 10, or 12, wherein the portioncomprises a fusion junction between RAF1 and its fusion partner and thefusion protein has RAF1 kinase activity.
 91. The molecule of claim 89,wherein the molecule is an oligonucleotide that hybridizes understringent conditions to: a) a fragment of SEQ ID NO:1 comprisingnucleotides 866-875 of SEQ ID NO:1; b) a fragment of SEQ ID NO:3comprising nucleotides 1693-1702 of SEQ ID NO:3; c) a fragment of SEQ IDNO:5 comprising nucleotides 3041-3050 of SEQ ID NO:5; d) a fragment ofSEQ ID NO:7 comprising nucleotides 1265-1274 of SEQ ID NO:7; e) afragment of SEQ ID NO:9 comprising nucleotides 143-152 of SEQ ID NO:9;f) a fragment of SEQ ID NO:11 comprising nucleotides 971-980 of SEQ IDNO:11; or g) a complementary oligonucleotide of any one of a)-f). 92.The molecule of claim 89, wherein the molecule is an antibody thatspecifically binds to: a) a fragment of SEQ ID NO:2 comprising aminoacids 286-295 of SEQ ID NO:2; b) a fragment of SEQ ID NO:4 comprisingamino acids 562-572 of SEQ ID NO:4; c) a fragment of SEQ ID NO:6comprising amino acids 1011-1020 of SEQ ID NO:6; or d) a fragment of SEQID NO:8 comprising amino acids 419-428 of SEQ ID NO:8; e) a fragment ofSEQ ID NO:10 comprising amino acids 45-54 of SEQ ID NO:10; or f) afragment of SEQ ID NO:12 comprising amino acids 321-330 of SEQ ID NO:12.